结核杆菌DNA探针板杂交方法及其应用研究

Microplate Hybridization of Mycobacterium Tuberculosis whole Chromosomal DNA Probe

我们建立了一种在酶标板上进行ＤＮＡ：ＤＮＡ杂交的方法，该方法与ＥＬＩＳＡ类似，简单、易操作、可半定量，便于临床实验室推广应用。对其杂交条件及影响因素进行了详细的探讨。通过该方法，地高辛标记的结核分支杆菌全染色体ＤＮＡ探针和１２种分支杆菌、７种非分支杆菌个染色体ＤＮＡ的杂交百分率有显著差别，除卡介苗、堪萨斯分支杆菌杂交百分率超过３０％，其余受试菌株均低于１０％。结果表明在酶标板上进行核酸杂交是完全可行的，利用已知细菌ＤＮＡ定量包被板，与未知的带标记的细菌ＤＮＡ杂交，可达到菌种鉴定的目的。

We have developed a simple microplate hybridization method with a M.tuberculosis whole chromosomal DNA probe,and researched its conditions and influential factors of hybridization.This method is similar to ELISA,carried out easilier,and thus can be easily used in routine laboratories.By this method,hybridization percent of M.tuberculosis is notably different from that of the other mycobacteria and nonmycobacteria tested,except that of BCG and M.kansasii is higher than 30%,the others is lower than 10%.Hence.it is feasible to conduct the nucleic acid hybridization in the microplate.lf the microplate was coated with known mycobacteral DNA,and then hybridized with unknown labelled mycobacteral DNA,we will be able to conduct mycobacteral species identification.